Lipoproteins are the primary carriers of plasma cholesterol. They are micellar lipid-protein complexes (particles) having a surface film, comprised of one or more proteins associated with polar lipids, that surround a cholesterol-containing core. Lipoproteins were originally classified based on their buoyant densities as measured by ultracentrifugation. Accordingly, four major density classes have been recognized: chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL).
Studies have established a direct correlation between plasma LDL cholesterol levels and the risk of coronary artery disease (CAD). That is, elevated levels of plasma cholesterol found in LDL particles correlate with an increased risk of CAD. Similarly, many studies have now shown that elevated plasma levels of lipoprotein(a), a component of LDL, evidence a high risk factor for heart disease and atherosclerosis.
The structure of the lipoprotein(a) molecule has been determined to comprise a protein portion identified as apolipoprotein B-100 linked through disulfide bonds to two apolipoprotein(a) molecules. The structure of lipoprotein(a) is similar to that of plasminogen, a blood component involved in vessel wound repair which is activated by proteolytic cleavage at a specific activation site by tissue-type plasminogen activator (t-PA) or urokinase. The region encompassing the plasminogen activation site ("activation site region") differs in amino acid sequence the analogous apolipoprotein(a) region. The sequence around the plasminogen activation site (arginine-valine) is (SEQ ID NO:5) LYS-CYS-PRO-GLY-ARG-VAL-VAL-GLY-GLY, whereas the analogous apolipoprotein(a) sequence is (SEQ ID NO:6) LYS-CYS-PRO-GLY-SER-ILE-VAL-GLY-GLY. See Eaton et al., Proc. Natl. Acad. Sci. USA, 84: 3224-3228, 3227 (1987), the disclosure of which is incorporated herein. Moreover, while plasminogen is activated by cleavage by urokinase or t-PA at arginine 560, apolipoprotein(a) may be inactive or not activatable by streptokinase, urokinase or t-PA. Id. For purposes of this disclosure, the above-described region of apolipoprotein(a) that is analogous to the activation site region of plasminogen will be hereinafter referred to as "the activation site region of apolipoprotein(a)."
Despite these differences in the plasminogen and analogous apolipoprotein(a) activation site regions, the amino acid sequence of lipoprotein(a) and plasminogen are considerably homologous, each having long, recurring sequences of amino acids called "kringles." Previous attempts to prepare lipoprotein(a) assays have utilized antibodies raised against the entire lipoprotein(a) molecule. The apolipoprotein(a) can be purified by known centrifugation methods, and the purified protein is then injected into animals to raise antibodies. Because of the considerable structural homology between lipoprotein(a) and plasminogen, most of the antibodies raised against the entire lipoprotein(a) molecule have recognized epitopes common to both lipoprotein(a) and plasminogen, i.e., the antibodies cross react with plasminogen. Consequently, such assays have met with limited success because of the low number of antibodies which do not cross react with plasminogen, and because of the uncertainty regarding what those few antibodies which do not cross react are binding to. Further, the ability to perform assays for apolipoprotein(a) have been hampered by the lack of standards against which assay results can be accurately compared.
Thus, it has not been easy to directly assay a serum sample for lipoprotein(a) because the plasminogen present in the serum yields a false positive result. For this reason, there is a continuing need for means to determine the presence or absence of lipoprotein(a) in a serum sample without the problem of false positive results due to plasminogen, and a continuing need for a standard against which assay results can be accurately measured.